Fig 1: BRMS1 overexpression inhibits invasion and migration induced by TRIM7 in osteosarcoma cell lines. (a) Transwell and (b) western blot analysis showing the effect of pLVX-Puro-TRIM7 and/or pLVX-Puro-BRMS1 lentivirus transduction on the invasion, migration, and expression of BRMS1, Vimentin, Twist1 and E-cadherin in SAOS2 cells. Scale bar: 50 µm. (c) After 21 days of intravenous injection with SAOS2 cells that have been transduced with lentivirus expressing TRIM7 and/or BRMS1, a histological inspection was measured by H&E. Scale bar: 200 µm. (d) Quantification of lung microscopic nodules and (e) incidence of metastasis in mice of each group (n = 11). All the experiments were repeated at least three times, and data are represented as mean ± SD. (a, b) ***P 0.001 (two-way ANOVA followed by Bonferroni post-tests) compared with the vector group. (a, b) ###P < 0.001 (two-way ANOVA followed by Bonferroni post-tests) compared with the TRIM7 group. (d) ***P < 0.001 (one-way ANOVA followed by Bonferroni post-tests) compared with the vector group. (d) ###P < 0.001 (one-way ANOVA followed by Bonferroni post-tests) compared with the TRIM7 group.
Fig 2: Correlation analyses in osteosarcoma samples. (a–c) Western blot analysis showing the expression of TRIM7 or BRMS1 expression in adjacent nontumorous (n = 6) and osteosarcoma samples (n = 10). ***P < 0.001 (Mann–Whitney U test) compared with adjacent nontumorous tissues (N). (d) Pearson correlation scatter plots in osteosarcoma tissues (n = 10). OS, osteosarcoma. All the experiments were repeated at least three times, and data are represented as mean ± SD..
Fig 3: TRIM7 interacts with BRMS1 and ubiquitinates it. (a) Purification of the TRIM7 complex was carried out according to the procedure described in Materials and Methods. Proteins were separated on SDS-PAGE and stained with Coomassie Blue. (b) List of TRIM7-associated proteins identified by MS analysis. (c) HOS and MG63 cell lysates were subjected to IP with anti-TRIM7, anti-BRMS1 or control IgG antibody. (d) The subcellular localization of TRIM7 (red) and BRMS1 (green) was measured by immunofluorescence. Nuclei were stained with DAPI (blue) for reference. Scale bar: 50 µm. (e) qRT-PCR and Western blot analysis showing the effect of TRIM7 overexpression on endogenous BRMS1 levels in SAOS2 cells in the presence of 10 µM proteasome inhibitor (MG132) or DMSO. (f) IP and western blot showing the effect of TRIM7 overexpression on the ubiquitination of BRMS1 in SAOS2 cells. (g) 293T cells were cotransfected with the Flag-BRMS1 (WT) or Flag-mutant BRMS1 constructs (K8R, K69R or K184R) along with myc-TRIM7 and His-Ub constructs, and the pull-down assay was carried out. All the experiments were repeated at least three times, and data are represented as mean ± SD.
Fig 4: TRIM7 regulates osteosarcoma cells’ response to chemotherapy by targeting BRMS1. CCK-8 showing the effect of pLKO.1-shTRIM7 and/or pLKO.1-shBRMS1 lentivirus transduction on the proliferation of MG63 cells treated with (a) 5 µg/ml ADR or (b) 2 ng/ml MTX. CCK-8 showing the effect of pLVX-Puro-TRIM7 and/or pLVX-Puro-BRMS1 lentivirus transduction on the proliferation of SAOS2 cells treated with (c) 5 µg/ml ADR or (d) 2 ng/ml MTX. All the experiments were repeated at least three times, and data are represented as mean ± SD. ***P < 0.001 (two-way ANOVA followed by Bonferroni post-tests) compared with the shNC or the vector group. ###P < 0.001 (two-way ANOVA followed by Bonferroni post-tests) compared with the shTRIM7 or the TRIM7 group.
Supplier Page from Abcam for Anti-BRMS1 antibody